PROJECT SUMMARY/ABSTRACT The pediatric solid tumor Ewing sarcoma remains an unmet clinical need, particularly for patients with metastatic or relapsed disease. It is a prototypical fusion oncoprotein-driven pediatric cancer with EWS-FLI1 present in the majority of Ewing sarcoma tumors. Analysis of EWS-FLI1 target gene promoters has revealed that EWS-FLI1 preferentially binds to repetitive GGAA-containing microsatellites in upregulated genes and reprograms gene regulatory circuits, acting as a pioneer factor. These data, in the context of the otherwise simple genomic landscape of Ewing sarcoma tumors, strongly suggest that epigenetic and transcriptional reprogramming are key mechanistic features of EWS-FLI-driven oncogenesis. Project 3 seeks to mechanistically dissect the core transcriptional circuitry in Ewing sarcoma tumors and to drug this aberrant transcriptional network with novel transcriptional inhibitors and degraders. In Aim 1, we will apply two approaches to identify the key transcriptional circuitry in this disease. Core regulatory circuits (CRC) define interconnected, feed forward auto-regulatory loops that establish the transcriptional program of a cell through their extended regulatory networks. CRCs are often hijacked to drive oncogenesis. Using ChIP-seq and RNAseq data generated from Ewing sarcoma cell lines, patient-derived xenografts (PDXs), and primary tumors, we will test the hypothesis that a CRC network exists in Ewing sarcoma tumors and that it can be therapeutically targeted. We will also take a complementary computational approach to identify master regulators (MRs) in Ewing tumors from RNAseq data and then test the hypothesis that the CRC/MR genes interact with EWS-FLI1 to regulate gene expression. In Aim 2, we will intersect the validated CRC/MR members with data from a genome-scale CRISPR-Cas9 screen in Ewing sarcoma cells to identify their critical CRC/MR dependencies. These dependencies will be comprehensively validated in vitro and in vivo with orthogonal CRISPR-Cas9, CRISPRi, and shRNA approaches. In Aim 3, we will target the aberrant transcriptional circuits in Ewing sarcoma tumors with combinations of clinically relevant transcriptional modulators and transcriptional inhibitors developed by the Chemistry/Structural Core and those emerging from Projects 1 and 2, including novel degraders of CDKs, BETs, and EWS-fusions. These combinations will be validated in PDX models of Ewing sarcoma developed by the Preclinical Models Core. Successful completion of the proposed studies will shed new light on the transcriptional networks that interacts with EWS-FLI1 and govern the maintenance of Ewing sarcoma, as well as validate therapeutic strategies for targeting the transcriptional circuitry in state-of-the-art preclinical models of Ewing sarcoma.